Malignant melanoma is usually resistant to drug therapy which historically has been non-selective in action and often very toxic. A novel approach is to target a specific defect found in melanoma cells. We and others have shown that exposure of melanoma cells to arginine deiminase (ADI), an enzyme that catalyzes the hydrolysis of arginine to citrulline, results in apoptotic cell death. This unique sensitivity to ADI is primarily due to the fact that melanoma cells, unlike normal cells, do not express argininosuccinate synthetase (ASS) and hence are unable to synthesize arginine. Transfection of ASS cDNA confers resistance to AD1, further confirming that lack of ASS expression is critical for ADI sensitivity. We formulated a pegylated form of ADI (ADI-PEG20) to reduce immunogenicity and to increase the half-life. ADI-PEG20 has shown significant antitumor activity in vivo with low toxicity. We have completed a Phase I trial of ADI-PEG20 in advanced melanoma. Remarkably, 5/10 patients had partial response when treated at a dose >160 IU/m2, a dose that depleted plasma arginine to non detectable levels for >7 days. No > grade 2 toxicity was observed. Interestingly, two patients who did not respond had ASS expression in their tumors. In this application, we plan to conduct a Phase II trial to confirm the antitumor activity in advanced melanoma as outlined in specific aim 1. In specific aim 2, we will assay ASS in tumor samples by immunohistochemistry and RT-PCR prior and after treatment to assess whether ASS expression can be a predictor for tumor response, and whether de-repression of ASS occurs at relapse. In specific aim 3, we will investigate the possible mechanism of apoptotic cell death by ADI-PEG20. In addition, the possible mechanism(s) of resistance will be examined by using an in-vitro cell line made resistant to ADI-PEG20 and by using de-novo resistant cell lines derived from tumors at time of treatment failure. In order to optimize future use of ADI-PEG20, we will investigate whether pharmacological manipulation can induce/repress ASS expression. Our goal is to improve the treatment outcome of melanoma while minimizing toxicity by targeting a specific defect in melanoma cells.